Cytomegalovirus (CMV) is a nearly ubiquitous but usually asymptomatic human pathogen in adults. However, primary infections neonatally, or of a fetus during pregnancy, often produces serious CNS sequelae. Reactivated infections are also a major cause of morbidity in AIDS patients and bone marrow, renal and heart transplant patients. The nucleotide analogue ganciclovir (DHPG) is the only effective therapeutic agent available at present to combat severe CMV infections, although its usefulness is limited. DNA viruses provide useful and important model systems for basic research into mechanisms of gene regulation and DNA synthesis in mammalian cells. CKV is the largest and most complex of human viruses. However, a comprehensive picture of the genetic structure of human CMV has recently become available as a result of the complete determination of the DNA sequence of its 229-kb genome. Consequently, many new insights into the molecular biology and pathogenicity of the virus are expected within the next few years. In situ hybridization procedures are also beginning to yield new information about the sites of persistent and latent infection and the extent of viral gene expression in vivo. We have recently identified a lytic cycle DNA replication origin (ori-U) in the unique L-segment of human and simian CMV genomes by a novel procedure using DHPG-induced chain termination. Using this information and a transient DNA-transfection system developed for identifying the essential replication genes in HSV and EBV, we propose to (1) Examine the structure and directionality of replicating HCMV DNA growth forks in the presence and absence of DHPG; (2) Further define and characterize essential cis-acting elements and cellular factor DNA-binding sites within ori-U; (3) Identify all of the HCMV genes required to replicate ori-U by transient cotransfection assays; (4) Ask whether HCMV encodes a unique origin-specific DNA-binding protein and define its consensus sequence recognition properties.